2008 Annual Meeting Abstracts
Development of AAV2-VEGF for Gene Therapy- In Vitro Kinetics of Gene Expression
Paul Liu, MD, Xiao Tian Wang, MD, Qiang Zhong Ma, PhD, Anthony Bais, PhD.
Roger Williams Medical Center, Providence, RI, USA.
Objective: Gene therapy for use in accelerating wound healing require the development of new vectors with a safe and effective profile and minimal morbidity. We reported in vivo data demonstrating efficacy and minimal morbidity using AAV2-bFGF to salvage ischemic tissue in a rat flap model. The objective of the current study was to produce and test efficicency of the AAV2 vector harboring the VEGF gene as our ongoing effects to tranfter VEGF gene into ischemic flaps.
Design and Methods: Virions encoding VEGF-165 and the marker GFP were generated using a helper-free triple transfection process. Purification was followed by establishment of viral particle titre using quantitative PCR. MOI was determined using flow cytometry. HEK293 cells were targeted in vitro with MOI of vectors at 0, 10, 50, and 100, respectively, and cultured for 0, 3, 14, and 21 days.
Outcome Measures: ELISA and immunofluorescent microscopy were used to document transgene expression of VEGF and GFP.
Results: Expression of VEGF-165 transgene peaked at 14 days and continousely expressed at higher levels at 21 days at all MOIs tested. The density of positive GFP expression increased significantly when the MOI was increased from 10 to 50. When MOI was 50 and 100, the density of GFP-positive cells were not significantly different.
Conclusions: We demonstrate successful construction of an AAV2-VEGF vector suitable for work in vivo. Because optimal effects of pro-angiogenic gene therapy may require more than one transgene product to be expressed simultaneously, the current work also shows the feasibility of two transgenes being driven by a single promoter.